Method, polypeptides, nucleotide sequence of XOR-6, a vitamin D-like receptor from xenopus

ABSTRACT

In accordance with the present invention, there are provided new members of the steroid receptor superfamily of receptors, a representative member of which has been designated XOR-6. Invention receptors are responsive to hydroxy, mercapto or amino benzoates, and are expressed, for example, in  Xenopus laevis  embryos. XOR-6 is most closely, although distantly, related to the vitamin D3 receptor (VDR). The proteins are about 73% identical in amino acid sequence in the DNA-binding domains and about 42% identical in the ligand binding domain. Like VDR, XOR-6 has an extended D region between the DNA and ligand binding domains. Notably, the region amino-terminal to the XOR-6 DNA-binding domain is extremely acidic. This may influence its ability to activate target genes. XOR-6 is not restricted to Xenopus because southern blots show the presence of XOR-6-related sequences in a variety of other vertebrates. Indeed, a human genomic clone for an XOR-6 related gene has recently been isolated. In accordance with a particular aspect of the present invention, there are also provided nucleic acid sequences encoding the above-identified receptor, as well as constructs and cells containing same, and probes derived therefrom. Furthermore, we have also discovered that hydroxy, mercapto or amino benzoates modulate the transcription activating effects of invention receptors.

This application is a filing under 35 U.S.C. §371 from PCT/US96/00058,filed Jan. 16, 1996; which is a continuation-in-part and claims priorityto U.S. patent application Ser. No. 08/374,445; filed Jan. 17, 1995, nowabandoned.

FIELD OF THE INVENTION

The present invention relates to intracellular receptors, and ligandstherefor. In a particular aspect, the present invention relates tomethods for the modulation of processes mediated by invention receptors,as well as methods for the identification of compounds which effect suchmodulation.

BACKGROUND OF THE INVENTION

Nuclear receptors constitute a large superfamily of ligand-activatedtranscription factors. Members of this family influence transcriptioneither directly, through specific binding to the promoters of targetgenes (see Evans, in Science 240:889-895 (1988), or indirectly, viaprotein-protein interactions with other transcription factors (see, forexample, Jonat et al., in Cell 62:1189-1204 (1990), Schuele et al., inCell 62:1217-1226 (1990), and Yang-Yen et al., in Cell 62:1205-1215(1990)). The steroid/thyroid receptor superfamily includes receptors fora variety of hydrophobic ligands including cortisol, aldosterone,estrogen, progesterone, testosterone, vitamin D₃, thyroid hormone andretinoic acid, as well as a number of receptor-like molecules, termed“orphan receptors” for which the ligands remain unknown (see Evans,1988, supra). These receptors all share a common structure indicative ofdivergence from an ancestral archetype.

Identification of ligands for orphan receptors presents a significantchallenge for the future since the number of orphan receptors which havebeen identified far exceeds the number of receptors with known ligands.Indeed, at least 40 genes, both vertebrate and invertebrate, have beenidentified which are structurally related to the steroid/thyroidreceptor superfamily, but whose ligands are unidentified. Among theseare Drosophila genes of known developmental significance including: thegap gene, knirps (Nauber et al., in Nature 336:489-492 (1988), theterminal gene tailless, involved in patterning the head and tail regions(Pignoni et al., in Cell 62:151-163 (1990), seven-up, which influencesphotoreceptor cell-fate (Mlodzik et al., in Cell 60: 211-224 (1990), andultraspiracle, a gene required both maternally and zygotically forpattern formation (Oro et al., in Nature 347: 298-301 (1990)).

The identification of important Drosophila developmental genes asmembers of the steroid/thyroid hormone receptor superfamily suggeststhat vertebrate orphan receptors will have important developmentalfunctions. Furthermore, the identification of ligands for orphanreceptors could lead to the discovery of novel morphogens, teratogensand physiologically important hormones.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have identified new membersof the steroid receptor superfamily of receptors, a representativemember of which has been designated XOR-6. Invention receptors areresponsive to hydroxy, mercapto or amino benzoates, and are expressed,for example, in Xenopus laevis embryos. XOR-6 is most closely, althoughdistantly, related to the vitamin D3 receptor (VDR). The proteins areabout 73% identical in amino acid sequence in the DNA-binding domainsand about 42% identical in the ligand binding domain. Like VDR, XOR-6has an extended D region between the DNA and ligand binding domains.Notably, the region amino-terminal to the XOR-6 DNA-binding domain isextremely acidic. This may influence its ability to activate targetgenes. XOR-6 is not restricted to Xenopus because southern blots showthe presence of XOR-6-related sequences in a variety of othervertebrates. Indeed, a human genomic clone for an XOR-6 related gene hasrecently been isolated.

In accordance with a particular aspect of the present invention, thereare also provided nucleic acid sequences encoding the above-identifiedreceptors, as well as constructs and cells containing same, and probesderived therefrom. Furthermore, we have also discovered that hydroxy,mercapto or amino benzoates modulate the transcription activatingeffects of invention receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a schematic comparison between XOR-6 and the humanvitamin D3 receptor. The two amino acid sequences were aligned using theprogram GAP (see Devereaux et al., in Nucl. Acids Res. 12:387-395(1984)). Similarity between XOR-6 and hVDR is expressed as percent aminoacid identity.

FIG. 2 demonstrates that XOR-6 and hRXRα interact in vivo. The plasmidsindicated in the figure were co-transfected into CV-1 cells along withthe reporter tk(galp)3-luc and CMX-βgal. Note the strong suppression ofbasal transcription when GAL-XOR6 was added (right panel). This ischaracteristic of previously characterized ligand-dependent RXRheterodimeric partners.

FIG. 3 illustrates the activation of XOR-6 by a variety of aminobenzoate derivatives. Thus, 10⁻⁶M of each compound was tested in theco-transfection assay for its ability to activate GAL-XOR6. Comparableresults were obtained with full-length XOR-6.

FIG. 4 illustrates the interaction of XOR-6 and RA signalling pathways,specifically demonstrating the synergism between partially purifiedXOR-6 agonist and the RXR ligand 9-cis RA. Receptors were transfectedinto cells and incubated with the indicated concentrations of agonists.

FIG. 5 illustrates the interaction of XOR-6 and RA signalling pathways,specifically demonstrating how the overexpression of full-length XOR-6,or the GAL-XOR-6 construct, interferes with retinoic acid (RA)signalling through the RARβ-RARE. 1 μg of XOR-6 expression plasmid wasco-transfected into CV-1 cells with 5 μg of tk-β REx2-luc, andchallenged with the indicated concentrations of all-trans retinoic acid.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have identified new membersof the steroid receptor superfamily of receptors, a representativemember of which has been designated XOR-6. Invention receptors areresponsive to hydroxy, mercapto or amino benzoates, and are expressed,for example, in Xenopus laevis embryos. Invention receptor comprises aprotein of approximately 386 amino acids (see SEQ ID NO:2), which ismost closely, although distantly, related to the vitamin D3 receptor(VDR). Also provided herein is a 2191 bp cDNA which encodes an exampleof invention receptors (see SEQ ID NO:1).

XOR-6 and VDR are about 73% identical in amino acid sequence in theDNA-binding domains and about 42% identical in the ligand bindingdomain. Like the VDR, XOR-6 has an extended D region between the DNA andligand binding domains. Notably, the region amino-terminal to the XOR-6DNA-binding domain is extremely acidic. This may influence its abilityto activate target genes. XOR-6 is not restricted to Xenopus becausesouthern blots show the presence of XOR-6-related sequences in a varietyof other vertebrates.

XOR-6 was discovered as part of a search for nuclear receptors expressedearly in Xenopus laevis development. Thus, cDNAs encoding transcriptsfrom nine different genes were isolated. These included xRARα, xRARγ,xRXRα, xRXRγ and five different orphan receptors. The presence of thisdiversity of receptors early in development suggests that their ligandsmight play important roles in morphogenetic signalling processes.Therefore it was of particular interest to identify those orphanreceptors which had a high probability of showing ligand dependence.

Because most known RXR heterodimeric partners are ligand responsive, theabove-described orphan receptor collection was screened for the abilityto heterodimerize with RXR both in vitro and in vivo. One such orphanreceptor, XOR-6 (for Xenopus Orphan Receptor 6). XOR-6 is a novelheterodimeric partner for RXR both in vitro and in vivo, furtherextending the family of nuclear receptors which require RXR forhigh-efficiency DNA-binding. XOR-6:RXR heterodimers apparently prefer tobind direct repeats separated by four nucleotides (DR-4), as does thethyroid hormone receptor. XOR-6 expression significantly blunts theability of RAR to activate gene expression suggesting that these twosignalling pathways block each other's ability to activate geneexpression perhaps by influencing their common heterodimeric partner,RXR.

Based on the presumption that XOR-6 and its ligand must be co-expressedat some time during development, an unbiased, bioassay directed screenfor XOR-6 agonists in HPLC fractionated organic extracts derived from amixture of developmental stages was undertaken. A potent agonist waspurified, and identified as 3-amino-ethyl-benzoate (3-AEB). Specificbinding of 3-AEB to XOR-6 has been demonstrated herein, identifying itas a true ligand for this receptor. Additional ligands for XOR-6, e.g.,hydroxy benzoates and mercapto benzoates, have also been identified.Accordingly, XOR-6 and ligands therefor represent a hitherto unknownhormonal signalling pathway.

RNAse protection assays were employed to measure steady-state mRNAlevels over a developmental time sequence. XOR-6 mRNA is present in theunfertilized egg and remains at a relatively constant level until aftergastrulation. It persists thereafter at a much reduced level until atleast stage 45. To investigate whether XOR-6 mRNA is localized in thepre-midblastula embryo, blastulae were dissected into three majorcomponents, the animal cap, marginal zone and endoderm. RNAse protectionanalysis showed that there is no obvious localization of the maternallyencoded XOR-6 mRNA at this stage.

Zygotic transcripts first become noticeable during neurulation (stage14) where they appear in the anterior neural folds and the regionlateral thereto. As the neural folds close, staining becomes more medialuntil finally appearing as an inverted Y at about stage 20. This isexactly the same pattern as cells which give rise to the hatching gland.Interestingly, this staining pattern defines boundaries of the futurehead. By stage 38, XOR-6 mRNA is restricted to the head, but is notlimited to the hatching gland.

In vitro DNA-binding studies were used to determine the DNA-bindingspecificity of XOR-6. XOR-6 and hRXRα are seen to heterodimerize andbind DNA in a cocktail of response elements. This binding is stronglycooperative, as neither receptor alone showed DNA-binding at the proteinconcentrations used in the assay. This binding is also specific tohRXRα, because hRARα does not enhance XOR-6 DNA binding. Similar resultsare obtained using xRXRα.

A finer analysis of XOR-6:hRXRα binding specificity shows that theheterodimer binds to a subset of the known response elements in thecocktail: it binds weakly to DR-3 (but not the osteopontin vitamin Dresponse element (SPP-VDRE), which is a variant of DR-3), strongly toDR-4 (and the murine leukemia virus (MLV-TRE), a DR-4-like element), andweakly to DR-5 (but strongly to the RARβ response element, a DR-5-likeelement). No significant binding is seen to synthetic or naturalresponse elements corresponding to DR-0,1,2 or 6 (i.e., direct repeatshaving spacers of 0, 1, 2 or 6 nucleotides, respectively). These dataindicate that the XOR-6:hRXRα heterodimer prefers to bind a DNA sequenceconsisting of directly-repeated AGTTCA half sites, separated by fournucleotides.

It was next tested to determine whether the XOR6:xRXRα heterodimerexhibited the predicted DNA-binding specificity. In vitro transcribed,translated XOR-6 and xRXRα proteins were tested for binding to directrepeats of AGTTCA separated by 1, 2, 3, 4, or 5 nucleotides (seePerlmann et al., in Genes Dev. 7:1411-1422 (1993)). The heterodimer isobserved to exhibit the expected binding specificity to a responseelement comprising two half-sites (each having the sequence AGTTCA)separated by 4 nucleotides. This allowed the design of a specific XOR-6reporter gene, tk-X6RE-luc (wherein the response element has thesequence AGTTCA TGAG AGTTCA; SEQ ID NO:3), which can be activated byXOR-6 in the presence of HPLC-purified embryo extracts.

In order to demonstrate that XOR-6 and RXR interact in vivo, amodification of the two hybrid system (see Fields and Song, in Nature340:245-246 (1989), or Nagpal et al., in Cell 70:1007-1019 (1992)) wasemployed. This system relies on functional dimeric interactions betweentwo proteins, one carrying the ability to bind a particular DNA-responseelement, and the other carrying the transactivation function, toreconstitute DNA-binding and transcriptional activation in a singlecomplex.

Applying this system to XOR-6 and RXR, VP16-hRXRα (a constitutiveactivator), GAL-XOR-6 and tk(gal_(p))₃-luc were employed. Functionalinteraction between XOR-6 and hRXRα should lead to constitutiveactivation of the reporter gene when all three constructs aretransfected together. VP16-hRXRα alone does not activate the reporterbecause it lacks the ability to bind to a GAL4 response element.Activation of the reporter occurs only when GAL-XOR-6 and VP16-hRXRα arecotransfected. Moreover, GAL-XOR-6 shows strong suppression of reportergene basal activity (gee FIG. 2), which parallels effects elicited byGAL-hRARα, GAL-hTRβ and GAL-hVDR. Based on these observations, it can beconcluded that XOR-6 and hRXRα can form functional heterodimers in vivo,that GAL-XOR-6 is unable to activate target genes in the absence of itsligand, and that unliganded GAL-XOR6, like most other ligand-dependentRXR partners, suppresses basal activity of a reporter construct to whichit can bind.

To demonstrate that XOR-6 hormone responsiveness differs from that ofother RXR dimeric partners (e.g., RAR, VDR, TR, and PPAR), the responseof GAL-XOR-6 to agonists for the above receptors was tested. GAL-XOR-6was not activated by a cocktail containing thyroid hormone (10⁻⁷M),vitamin D3 (10⁻⁷M), all-trans RA (10⁻⁶M), or the peroxisome proliferatorWY-14,643 (5×10⁻⁶M), while GAL-VDR, GAL-hRARα, GAL-hTRβ, and GAL-mPPARαare activated by the cocktail. It can be concluded, therefore, thatXOR-6 defines a novel RXR-dependent, ligand-mediated signalling pathway.

A search for the XOR-6 ligand was instituted based on the presumptionthat the receptor and its ligand must be co-expressed at some timeduring development. Accordingly, an unbiased, bioassay directed screenfor XOR-6 agonists was undertaken in HPLC fractionated organic extractsderived from a mixture of developmental stages. Total lipid extractsfrom a mixture of embryonic stages from fertilized eggs through swimmingtadpoles were prepared and tested for the ability to activate bothGAL-XOR6 or full-length XOR-6 in transfected CV-1 cells.

The total extract was partitioned between iso-octane and MeOH and againtested for bioactivity. Since the methanol phase contained most of theactivity, it was further partitioned between ethyl acetate and H₂O. Theethyl acetate phase was shown to contain most of the activity and wasthus further purified by reverse phase HPLC using several solventsystems. Absorbance was monitored between 200 and 600 nm, fractions werecollected, dried and tested in the cotransfection assay (see, forexample, U.S. Pat. No. 5,071,773) for their ability to activatefull-length and GAL-XOR6. The eluted, purified agonist was subjected tohigh resolution mass spectroscopy which yielded a mass/charge ratio of165.19 daltons. This predicted a molecular formula of C₉H₁₁O₂N, whichmost closely matches the ethyl ester of amino benzoic acid (AEB). Thefragmentation pattern in Electron Impact mass spectroscopy suggests themeta isomer of AEB as the predominant form.

The ortho, meta and para amino ethyl benzoates were tested for agonistactivity in the cotransfection assay. All three activated XOR-6 with arank order potency as follows:

3-AEB>4-AEB>>2-AEB.

3-AEB co-chromatographed with purified agonist and gave an identical UVspectrum to authentic 3-AEB. Thus, 3-AEB is unequivocally identified asthe purified agonist. Moreover, 3-AEB specifically activates XOR-6 aloneamong an extensive collection of published and unpublished vertebratenuclear receptors.

In order to investigate ligand binding, the protease protection assaydescribed by Leng et al., in J. Ster. Bioch. and Mol. Biol. 46:643-661(1993) and Keidel et al, in Mol. Cell. Biol. 14:287-298 (1994) wasutilized. Thus, ³⁵S-labelled in vitro transcribed translated protein wasincubated with increasing concentrations of various proteases in thepresence of solvent carrier or the putative ligand. The presence of3-AEB results in some protection from trypsin cleavage with aconcomitant increase in the intensity of the intermediately sizedcleavage products. This result is not seen in parallel experiments withxRARα or xRXRα, again suggesting specificity in ligand binding.

It was next attempted to determine whether compounds related to 3-AEBmight also function as ligand for invention receptor. One likelycandidate is the vitamin, 4-amino-benzoic acid (PABA). It was notpossible, however, to demonstrate XOR-6 activation by 2-, 3-, or 4-aminobenzoic acids, or the related 2-, 3-, or 4-amino salicylic acids. It ispossible that the cell membrane is much less permeable to the acids thanto the more lipophilic esters. This possibility was tested by comparingthe activation by a series of esters differing in the length of thealkyl group. As shown in FIG. 3, the more lipophilic esters showedincreased activation with a rank order potency of 4-amino-butylbenzoate>3-amino-ethyl benzoate>4-amino-ethyl benzoate>>4-amino methylbenzoate. These results suggest that the limiting step in XOR-6activation is the transport of the ligand through the cell membrane. Inconjunction with these studies, additional substituted benzoates, e.g.,hydroxy benzoates and mercapto benzoates, have also been identified asligands for invention receptor.

A potentially significant property of the XOR6:xRXRα heterodimer is itsresponsiveness to two ligands. Thus, in co-transfection experiments,either 9-cis RA or the partially purified agonist stimulated reportergene expression in a receptor dependent manner. Unlike the response ofRAR, VDR and TR heterodimers with RXR, which show additive effects ontranscription, the XOR-6 ligand synergizes with 9-cis retinoic acid toactivate its reporter gene (see FIG. 4), reminiscent of the situationwith PPAR (see Kliewer et al., in Nature 358:771-774 (1992)). Thissynergism occurs at several dilutions of the XOR-6 agonist andconcentrations of 9-cis RA (see FIG. 4). The demonstration of anotherheterodimer with dual hormone-responsiveness suggests that nuclearreceptor heterodimers can generate combinatorial diversity by creatingcomplexes with both novel DNA-binding properties and multiple hormonalactivation levels. Such complexes would be ideal candidates forresponding to combinations of graded morphogenetic signals duringdevelopment.

Because XOR-6:RXR heterodimers bind well to a retinoic acid responseelement, βRARE, it was tested whether overexpression of XOR-6 couldinfluence retinoic acid signalling through this element. As shown inFIG. 5, it is found that co-expression of XOR-6 and βRARE significantlyblunts the retinoic acid-responsiveness of this promoter in adose-dependent manner. This effect was strongest with full-length XOR-6(24% of wild-type activity) but still detectable with GAL-XOR-6 (44% ofwild-type activity). This suggests that maximal repression results frombinding of XOR-6:RXR heterodimers to the βRARE, producing anon-productive transcription complex. The weaker inhibition by GAL-XOR-6(which cannot bind to βRARE) suggests that sequestration of RXR inheterodimers unresponsive to retinoic acid also plays an inhibitoryrole.

In accordance with another embodiment of the present invention, thereare provided a class of hydroxy, mercapto or amino benzoate compoundswhich are capable of acting as ligands for invention receptors. Asemployed herein, the phrase “hydroxy, mercapto or amino benzoate(s)”embraces compounds having the structure:

wherein

X is an hydroxy, alkoxy (of a lower alkyl, i.e., having 1-4 carbonatoms), mercapto, thioalkyl (of a lower alkyl), amino, alkylamino oracylamino group at the 2-, 3-, or 4-position of the ring,

each Y, when present, is independently selected from hydroxy, alkoxy,mercapto, thioalkyl, halide, trifluoromethyl, cyano, nitro, amino,carboxyl, carbamate, sulfonyl, sulfonamide, and the like,

Z is selected from —OR′ or —NHR′, wherein R′ is selected from hydrogen,C₁-C₁₂ alkyl, or C₅-C₁₀ aryl, and

n is 0-2.

Presently preferred compounds embraced by the above generic formulainclude those wherein X is 2-, 3-, or 4-hydroxy or 3- or 4-amino, Z isalkoxy (i.e., methoxy, ethoxy or butoxy) and n is 0.

In accordance with yet another embodiment of the present invention,there are provided nucleic acids which encode the above-describedreceptor polypeptides. Exemplary DNAs include those which encodesubstantially the same amino acid sequence as shown in SEQ ID NO:2(e.g., a contiguous nucleotide sequence which is substantially the sameas nucleotides 166-1324 shown in SEQ ID NO:1). Preferred DNAs includethose which encode the same amino acid sequence as shown in SEQ ID NO:2(e.g., a contiguous nucleotide sequence which is the same as nucleotides166-1324 shown in SEQ ID NO:1).

As used herein, nucleotide sequences which are substantially the sameshare at least about 90% identity, and amino acid sequences which aresubstantially the same typically share more than 95% amino acididentity. It is recognized, however, that proteins (and DNA or mRNAencoding such proteins) containing less than the above-described levelof homology arising as splice variants or that are modified byconservative amino acid substitutions (or substitution of degeneratecodons) are contemplated to be within the scope of the presentinvention.

In accordance with still another embodiment of the present invention,there are provided DNA constructs comprising the above-described DNA,operatively linked to regulatory element(s) operative for transcriptionof said DNA and expression of said polypeptide in an animal cell inculture. There are also provided cells containing such construct,optionally containing a reporter vector comprising:

(a) a promoter that is operable in said cell,

(b) a hormone response element, and

(c) DNA encoding a reporter protein,

wherein said reporter protein-encoding DNA is operatively linked to saidpromoter for transcription of said DNA, and

wherein said promoter is operatively linked to said hormone responseelement for activation thereof.

In accordance with a still further embodiment of the present invention,there are provided probes comprising labeled single-stranded nucleicacid, comprising at least 20 contiguous bases in length havingsubstantially the same sequence as any 20 or more contiguous basesselected from bases 1-2150, inclusive, of the DNA illustrated in SEQ IDNO:1, or the complement thereof. An especially preferred probe of theinvention comprises at least 20 contiguous bases in length havingsubstantially the same sequence as any 20 or more contiguous basesselected from bases 473-1324, inclusive, of the DNA illustrated in SEQID NO:1, or the complement thereof.

Those of skill in the art recognize that probes as described herein canbe labelled with a variety of labels, such as for example, radioactivelabels, enzymatically active labels, fluorescent labels, and the like. Apresently preferred means to label such probes is with ³²P. Such probesare useful, for example, for the identification of receptorpolypeptide(s) characterized by being responsive to the presence ofhydroxy, mercapto or amino benzoate(s) to regulate the transcription ofassociated gene(s), said method comprising hybridizing test DNA with aprobe as described herein under high stringency conditions (e.g.,contacting probe and test DNA at 65° C. in 0.5 M NaPO₄, pH 7.3, 7%sodium dodecyl sulfate (SDS) and 5% dextran sulfate for 12-24 hours;washing is then carried out at 60° C. in 0.1×SSC, 0.1% SDS for threethirty minute periods, utilizing fresh buffer at the beginning of eachwash), and thereafter selecting those sequences which hybridize to saidprobe.

In another aspect of the invention, the above-described probes can beused to assess the tissue sensitivity of an individual to hydroxy,mercapto or amino benzoates by determining XOR-6 mRNA levels in a giventissue sample. It is expected that an individual having a high level ofXOR-6 mRNA (or protein) will be sensitive to the presence of significantlevels of amino benzoates, such as are used in sunscreen applications.

In accordance with yet another embodiment of the present invention,there are provided antibodies which specifically bind theabove-described receptor polypeptides. Preferably, such antibodies willbe monoclonal antibodies. Those of skill in the art can readily preparesuch antibodies having access to the sequence information providedherein regarding invention receptors.

Thus, the above-described antibodies can be prepared employing standardtechniques, as are well known to those of skill in the art, using theinvention receptor proteins or portions thereof as antigens for antibodyproduction. Both anti-peptide and anti-fusion protein antibodies can beused (see, for example, Bahouth et al. Trends Pharmacol Sci. 12:338-343(1991); Current Protocols in Molecular Biology (Ausubel et al., eds.)John Wiley and Sons, New York (1989)). Factors to consider in selectingportions of the invention receptors for use as immunogen (as either asynthetic peptide or a recombinantly produced bacterial fusion protein)include antigenicity, uniqueness to the particular subtype, and thelike.

The availability of such antibodies makes possible the application ofthe technique of immunohistochemistry to monitor the distribution andexpression density of invention receptors. Such antibodies could also beemployed for diagnostic and therapeutic applications.

In accordance with yet another embodiment of the present invention,there is provided a method of testing a compound for its ability toregulate transcriptionactivating effects of invention receptorpolypeptide(s), said method comprising assaying for the presence orabsence of reporter protein upon contacting of cells containing saidreceptor polypeptide and reporter vector with said compound;

wherein said reporter vector comprises:

(a) a promoter that is operable in said cell,

(b) a hormone response element, and

(c) DNA encoding a reporter protein,

wherein said reporter protein-encoding DNA is operatively linked to saidpromoter for transcription of said DNA, and

wherein said promoter is operatively linked to said hormone responseelement for activation thereof.

Hormone response elements suitable for use in the above-described assaymethod comprise two half sites (each having the sequence AGTTCA),separated by a spacer of 3, 4 or 5 nucleotides. Those of skill in theart recognize that any combination of 3, 4 or 5 nucleotides can be usedas the spacer. Response elements having a spacer of 4 nucleotides (e.g.,SEQ ID NO:3) are presently preferred.

Optionally, the above-described method of testing can be carried out inthe further presence of ligand for invention receptors (e.g., a hydroxy,mercapto or amino benzoate), thereby allowing the identification ofantagonists of invention receptors. Those of skill in the art canreadily carry out antagonist screens using methods well known in theart. Typically, antagonist screens are carried out using a constantamount of agonist, and increasing amounts of a putative antagonist.

In accordance with a still further embodiment of the present invention,there is provided a method for modulating process(es) mediated byinvention receptor polypeptides, said method comprising conducting saidprocess(es) in the presence of at least one hydroxy, mercapto or aminobenzoate (as defined hereinabove).

As shown herein, XOR-6 and RXR functionally interact both in vitro topreferentially bind a DR-4 type response element, and in vivo toactivate a GAL4-based reporter in the two-hybrid assay. Thus afunctional interaction has been identified between RXR and an orphanreceptor within the cell to activate a reporter gene. This observationcan be exploited to develop a high-sensitivity assay system for theXOR-6 ligand and for orphan receptor ligands in general, at least forthose which interact with RXR.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 cDNA Isolation and Characterization

XOR-6 was identified in a screen for maternally-expressed nuclearhormone receptors (Blumberg et al., in Proc. Natl. Acad. Sci. USA89:2321-2325 (1992). Three clones were identified from an egg cDNAlibrary, an additional two were isolated from a dorsal blastopore lipcDNA library. The longest clone was sequenced completely on both strandsusing a combination of directed subcloning and specific oligonucleotidepriming. DNA sequences were compiled and aligned using the programs ofStaden (Staden, in Nucleic Acids Res. 14:217-231 (1986), University ofWisconsin Genetics Computer Group (Devereaux et al., 1984, supra, andFeng and Doolittle (Feng and Doolittle, in J. Mol. Evol. 215:351-260(1987). Database searching was performed using the BLAST network serverat the National Center for Biotechnology Information (Altschul et al.,J. Mol. Biol. 215:403-410 (1990)).

EXAMPLE 2 RNA Preparation and Analysis

RNA was prepared from fertilized Xenopus laevis eggs and staged embryosas described by Blumberg et al., 1992, supra. The temporal and spatialpatterns of expression were determined using RNAse protection asdescribed by Blumberg et al., 1992, supra. The RNAse protection probesused are the following: EF-1α, nucleotides 790-1167; XOR-6, nucleotides1314 to 1560, which represents the last three amino acids of the proteinand part of the 3′ untranslated region.

RNAse protection was performed with total RNA from the total ovary (10μg); unfertilized egg (40 μg); 2-cell (40 μg); blastula (40 μg);gastrula (st 10, 10 μg), st 11, 8 μg); neurula (4 μg); tailbud (4 μg);swimming tadpole (4 μg). Alternatively, RNAse protection was performedwith 20 μg of total RNA from whole embryos or dissected animal caps,marginal zone, and vegetal pole.

A lateral view of a stage 12 embryo hybridized with antisense XOR-6reveals that hybridization extends from the anterior-most end of theinvoluting mesoderm to the dorsal blastopore lip.

For localization studies, stage 8-9 embryos were dissected into animal,marginal and vegetal fragments and RNA was prepared using a proteinase Kmethod as described by Cho et al., in Cell 65:55-64 (1991). Whole-mountin situ hybridization was performed as described by Harland, (1991). Theentire cDNA shown in SEQ ID NO:1 was used as a probe for in situhybridization. To make anti-sense RNA, the Bluescript II SK-plasmidcontaining the cDNA was linearized with SmaI and transcribed with T7 RNApolymerase. To produce sense RNA, the plasmid was digested with EcoRVand transcribed with T3 RNA polymerase.

EXAMPLE 3 In vitro DNA-binding

DNA-binding analysis was performed using in vitro transcribed,translated proteins (Perlmann et al., 1993, supra. oligonucleotidesemployed have been described previously (see Umesono et al., in Cell65:1255-1266 (1990) and Perlmann et al., 1993, supra).

Thus, in vitro transcribed and translated proteins were mixed with acocktail of hormone response elements containing DR0, DR1, PPRE, DR2,MLV-TRE, SPP1, and β-RARE. Thus, XOR-6 and hRXRα proteins were mixed andincubated with radiolabelled response elements. DR-1 through 5 aredirect repeats of the sequence AGTTCA separated by 1-5 nucleotides.Reaction conditions and gel electrophoresis employed were as describedby Perlmann et al., 1993, supra.

EXAMPLE 4 Cell Culture and Transfection Studies

A suitable eukaryotic expression vector for use herein was constructedfrom the commercially available vector pCDNAI-AMP (Invitrogen). Thisvector allows expression from the strong cytomegalovirus early promoter,and bacteriophage T7 and SP6 promoter-driven production of sense andantisense RNA, respectively.

The cloning strategy employed was as follows: the three endogenous NcoIsites were removed by site directed mutagenesis, the polylinker regionbetween XhoI and XbaI was removed by double digestion, endfilling andself ligation. A cassette consisting of the Xenopus β-globin leader andtrailer derived from the plasmid pSP36T (see Amaya et al., in Cell66:257-270 (1991)), separated by a synthetic polylinker (containingunique sites for NcoI, SphI, EcoRI, SalI, EcoRV, BamHI, and XbaI) wasinserted between HindIII and NotI sites in the vector. The resultingplasmid, designated pCDG1, can be linearized with NotI to produce mRNAfrom the bacteriophage T7 promoter. The XOR-6 protein coding region wascloned between the NcoI and BamHI sites of pCDG1 and designatedpCDG-XOR6.

pCMX-GAL4-XOR6 was constructed by cloning nucleotides encoding aminoacids 103 to 386 of XOR-6 into the SalI to XbaI sites of pCMX-GAL4 (seeU.S. Ser. No. 08/177,740).

pCMX-VP16 receptor chimeras were constructed by fusing the potent VP16transactivation domain (see Sadowski et al., in Nature 335:563-564(1988)) to the amino terminus of the full-length hRXRα (see Mangelsdorfet al., Nature 345:224-229 (1990)), hRARα (see Giguere et al., in Nature330:624-629 (1987)), or VDR (see McDonnell et al., in Mol. Endocrinol.3:635-644 (1989)) protein coding regions.

CV-1 cells were maintained in DMEM containing 10% resin-charcoalstripped fetal bovine serum. Liposome-mediated transient transfectionswere performed using DOTAP reagent (Boehringer Manheim) at aconcentration of 5 μg/ml in Opti-MEM (Gibco). After 12-18 hours, thecells were washed and fresh DMEM-10% serum was added, including receptoragonists if required. After a further 48 hour incubation, the cells werelysed and luciferase reporter gene assays and β-galactosidasetransfection control assays performed. Reporter gene expression isnormalized to the β-galactosidase transfection control and expressed asrelative light units per O.D. per minute of β-galactosidase activity.

EXAMPLE 5 Organic Extraction and HPLC Analysis

Fresh or flash frozen embryos were homogenized in a large volume of 50%CH₂Cl₂/50% MeOH, typically 10 ml/gram of tissue. Denatured proteins wereremoved by filtration through diatomaceous earth and the liquid phaserecovered and evaporated to dryness with a Buchi rotary evaporator. Theresulting material was resuspended in a minimum volume of iso-octane andtransferred to a separatory funnel. Non-polar and polar compounds wereseparated by partitioning between large volumes of iso-octane and MeOH.An agonist of XOR-6 partitioned primarily into the methanol layer.

The methanol phase was then dried, weighed, and partitioned betweenethyl acetate and H₂O. An agonist for XOR-6 partitioned greater than 95%into ethyl acetate. The ethyl acetate phase was then dried, weighed, andfractionated by reverse phase HPLC, using several solvent systems.

Initially, the ethyl acetate phase was separated by isocratic elutionutilizing a 7.8×300 mm Novapack C18 column (Waters), developed at 4ml/min with 56% acetonitrile, 16% methanol, 28% 2% aqueous acetic acid(see Heyman et al., in Cell 68:1-20 (1992)). Absorbance was monitoredbetween 200 and 600 nm using a Waters 996 photodiode array detector.Fractions were collected, dried and tested in the cotransfection assayfor their ability to activate GAL-XOR6. Active fractions were pooled andrechromatographed on the same column using a gradient of methanol, 10 mMammonium acetate (pH 7.5) beginning at 30% methanol, run isocraticallyfor 15 minutes, and then increasing linearly to 100% methanol over thenext 45 minutes. Fractions were again tested for bioactivity and theactive fractions pooled.

Final purification was accomplished using a dioxane/water gradientbeginning at 20% dioxane and run isocratically for 15 minutes, thenincreasing linearly to 100% dioxane over the next 30 minutes.

EXAMPLE 6 Ligand Binding

In order to investigate ligand binding, a protease protection assay wasutilized (see Leng et al., 1993, supra, and Keidel et al, 1994, supra).³⁵S-labelled protein was produced by coupled in vitrotranscription/translation (TNT, Promega) and incubated with increasingconcentrations of trypsin, chymotrypsin or alkaline protease in thepresence of solvent carrier or with 10⁻⁵M 3-amino ethylbenzoate (3-AEB)for 15 minutes at room temperature. The reactions were stopped withSDS-loading buffer and SDS-PAGE was performed on 12.5% acrylamide gels.Alterations in the size of protected fragments produced by added ligandin a dose dependent fashion was taken as evidence for specific binding.

3-AEB is seen to protect XOR-6 from trypsin digestion, thus confirmingthat 3-AEB binds XOR-6.

While the invention hag been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

That which is claimed is:
 1. A method for modulating transcriptionactivating effects mediated by nuclear receptor polypeptides whereinsaid receptor has substantially the same amino acid sequence as SEQ IDNO:2, or is encoded by a nucleic acid that hybridizes to a nucleic acidof SEQ ID NO:1 under high stringency conditions, said method comprisingconducting said transcription in the presence of one or more of saidreceptor polypeptides and a compound having the structure:

wherein X is a hydroxy, alkoxy, mercapto, thioalkyl, amino, alkylaminoor acylamino group at the 2-3-, or 4-position of the ring, each Y, whenpresent, is independently selected from hydroxy, alkoxy, mercapto,thioalkyl, halide, trifluoromethyl, cyano, nitro, amino, carboxyl,carbamate, sulfonyl, or sulfonamide, Z is selected from —OR′ or —NHR′,wherein R′ is selected from hydrogen, C₁-C₁₂ alkyl or C₅-C₁₀ aryl, and nis 0-2.
 2. A method according to claim 1, wherein said compound is atleast one hydroxy, mercapto or amino benzoate.
 3. A method according toclaim 1 wherein X is 3- or 4-amino, Z is alkoxy and n is
 0. 4. A methodaccording to claim 3 wherein Z is selected from methoxy, ethoxy orbutoxy.
 5. A method according to claim 1 wherein X is 2-, 3- or4-hydroxy, Z is alkoxy and n is
 0. 6. A method according to claim 5wherein Z is selected from methoxy, ethoxy or butoxy.
 7. A method oftesting a compound for its ability to regulate transcription-activatingeffects of a nuclear receptor polypeptide wherein said receptor hassubstantially the same amino acid sequence as SEQ ID NO:2, or is encodedby a nucleic acid that hybridizes to a nucleic acid of SEQ ID NO:1 underhigh stringency conditions, said method comprising assaying for a changein expression of reporter protein upon contacting of test cells withsaid compound, as compared to the expression of said reporter protein inthe absence of said compound, and identifying as a compound thatregulates the transcription-activating effects of said receptor thosewhich cause a change in expression of said reporter protein whencompared to the expression of said reporter in the absence of saidcompound; wherein said test cell comprises said receptor polypeptide andreporter vector, wherein said reporter vector comprises: (a) a promoterthat is operable in said test cell, (b) a hormone response element forsaid receptor, and (c) DNA encoding said reporter protein, wherein saidreporter protein-encoding DNA is operatively linked to said promoter fortranscription of said DNA, wherein said promoter is operatively linkedto said hormone response element for activation thereof by said receptorpolypeptide.
 8. A method according to claim 7, wherein said receptor hassubstantially the same amino acid sequence as SEQ ID NO:2.
 9. A methodaccording to claim 7, wherein said cells also express RXR.
 10. A methodaccording to claim 7, comprising conducting said transcription in thepresence of RXR.
 11. A method according to claim 7, wherein saidreceptor is further characterized as being responsive to the presence ofhydroxy, mercapto or amino benzoate(s) to regulate the transcription ofassociated gene(s), said hydroxy, mercapto or amino benzoate(s) arecompounds having the structure:

wherein: N is an amino, hydroxy or mercapto-substituted aryl moiety, andZ is —OR′ or —NHR′, wherein R′ is hydrogen, C₁-C₁₂ alkyl or C₅-C₁₀ aryl.12. A method according to claim 11, wherein said contacting is carriedout in the further presence of at least one of said hydroxy, mercapto oramino benzoate(s).
 13. A method of testing a compound for its ability toregulate transcription-activating effects of a nuclear receptorpolypeptide wherein said receptor is encoded by a nucleic acid that hassubstantially the same sequence as the nucleic acid of SEQ ID NO:1, saidmethod comprising assaying for a change in expression of reporterprotein upon contacting of test cells with said compound, as compared tothe expression of said reporter protein in the absence of said compound,and identifying as a compound that regulates thetranscription-activating effects of said receptor those which cause achange in expression of said reporter protein when compared to theexpression of said reporter in the absence of said compound; whereinsaid test cell comprises said receptor polypeptide and reporter vector,wherein said reporter vector comprises: (a) a promoter that is operablein said test cell, (b) a hormone response element for said receptor, and(c) DNA encoding said reporter protein, wherein said reporterprotein-encoding DNA is operatively linked to said promoter fortranscription of said DNA, wherein said promoter is operatively linkedto said hormone response element for activation thereof by said receptorpolypeptide.
 14. A method of testing a compound for its ability toregulate transcription-activating effects of a nuclear receptorpolypeptide wherein said receptor comprises a DNA binding domain havingat least about 73 percent homology with residues 37-102 of the aminoacid sequence of SEQ ID NO:2, said method comprising assaying for achange in expression of reporter protein upon contacting of test cellswith said compound, as compared to the expression of said reporterprotein in the absence of said compound, and identifying as a compoundthat regulates the transcription-activating effects of said receptorthose which cause a change in expression of said reporter protein whencompared to the expression of said reporter in the absence of saidcompound; wherein said test cell comprises said receptor polypeptide andreporter vector, wherein said reporter vector comprises: (a) a promoterthat is operable in said test cell, (b) a hormone response element forsaid receptor, and (c) DNA encoding said reporter protein, wherein saidreporter protein-encoding DNA is operatively linked to said promoter fortranscription of said DNA, wherein said promoter is operatively linkedto said hormone response element for activation thereof by said receptorpolypeptide.
 15. A method according to claim 14, wherein said receptoris further characterized by being responsive to the presence of hydroxy,mercapto or amino benzoate(s) and/or ester and/or imido derivativesthereof to regulate the transcription of associated gene(s).
 16. Amethod according to claim 14, wherein said receptor further comprises: aligand binding domain having at least about 42 percent homology toresidues 183-386 of the amino acid sequence of SEQ ID NO:2.
 17. A methodaccording to claim 16, wherein said receptor is further characterized bybeing responsive to the presence of hydroxy, mercapto or aminobenzoate(s) and/or ester and/or imido derivatives thereof to regulatethe transcription of associated gene(s).
 18. A method for modulatingtranscription activating effects mediated by nuclear receptorpolypeptides wherein said receptor is encoded by a nucleic acid that hassubstantially the same sequence as the nucleic acid of SEQ ID NO:1, saidmethod comprising conducting said transcription in the presence of oneor more of said receptor polypeptides and a compound having thestructure:

wherein X is a hydroxy, alkoxy, mercapto, thioalkyl, amino, alkylaminoor acylamino group at the 2-3-, or 4-position of the ring, each Y, whenpresent, is independently selected from hydroxy, alkoxy, mercapto,thioalkyl, halide, trifluoromethyl, cyano, nitro, amino, carboxyl,carbamate, sulfonyl, or sulfonamide, Z is selected from —OR′ or —NHR′,wherein R′ is selected from hydrogen, C₁-C₁₂ alkyl or C₅-C₁₀ aryl, and nis 0-2.
 19. A method for modulating transcription activating effectsmediated by nuclear receptor polypeptides wherein said receptorcomprises a DNA binding domain having at least about 73 percent homologywith residues 37-102 of the amino acid sequence of SEQ ID NO:2, saidmethod comprising conducting said transcription in the presence of oneor more of said receptor polypeptides and a compound having thestructure:

wherein X is a hydroxy, alkoxy, mercapto, thioalkyl, amino, alkylaminoor acylamino group at the 2-3-, or 4-position of the ring, each Y, whenpresent, is independently selected from hydroxy, alkoxy, mercapto,thioalkyl, halide, trifluoromethyl, cyano, nitro, amino, carboxyl,carbamate, sulfonyl, or sulfonamide, Z is selected from —OR′ or —NHR′,wherein R′ is selected from hydrogen, C₁-C₁₂ alkyl or C₅-C₁₀ aryl, and nis 0-2.
 20. A method according to claim 19, wherein said receptor isfurther characterized by being responsive to the presence of hydroxy,mercapto or amino benzoate(s) and/or ester and/or imido derivativesthereof to regulate the transcription of associated gene(s).
 21. Amethod according to claim 19, wherein said receptor further comprises: aligand binding domain having at least about 42 percent homology toresidues 183-386 of the amino acid sequence of SEQ ID NO:2.
 22. A methodaccording to claim 21, wherein said receptor is further characterized bybeing responsive to the presence of hydroxy, mercapto or aminobenzoate(s) and/or ester and/or imido derivatives thereof to regulatethe transcription of associated gene(s).